sign in sign up
<<
Home , Antimicrobial peptides, Lantibiotics, Surfactin

Small cationic antimicrobial delocalize PNAS PLUS peripheral membrane proteins

Michaela Wenzela, Alina Iulia Chiriacb, Andreas Ottoc, Dagmar Zweytickd, Caroline Maye, Catherine Schumacherf, Ronald Gustg, H. Bauke Albadah, Maya Penkovah, Ute Krämeri, Ralf Erdmannj, Nils Metzler-Nolteh, Suzana K. Strausk, Erhard Bremerl, Dörte Becherc, Heike Brötz-Oesterheltf, Hans-Georg Sahlb, and Julia Elisabeth Bandowa,1

aBiology of Microorganisms, hBioinorganic Chemistry, and iPlant Physiology, eImmune Proteomics, Medical Proteome Center, and jInstitute of Physiological Chemistry, Ruhr University Bochum, 44801 Bochum, Germany; bInstitute for Medical Microbiology, Immunology, and Parasitology, Pharmaceutical Microbiology Section, University of Bonn, 53113 Bonn, Germany; cDepartment of Microbial Physiology and Molecular , Ernst Moritz Arndt University, 17489 Greifswald, Germany; dBiophysics Division, Institute of Molecular Biosciences, University of Graz, 8010 Graz, Austria; fInstitute for Pharmaceutical Biology and Biotechnology, Heinrich Heine University, 40225 Düsseldorf, Germany; gDepartment of Pharmaceutical Chemistry, University of Innsbruck, 6020 Innsbruck, Austria; kDepartment of Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1Z1; and lDepartment of Biology, University of Marburg, 35037 Marburg, Germany

Edited by Michael Zasloff, Georgetown University Medical Center, Washington, DC, and accepted by the Editorial Board February 27, 2014 (received for review October 22, 2013)

Short rich in (R) and tryptophan synthetic hexapeptide RWRWRW-NH2 (referred to hereafter as (W) interact with membranes. To learn how this interaction leads “MP196”) (see SI Appendix, Fig. S1 for structure) as a model to bacterial death, we characterized the effects of the minimal representing the minimal pharmacophore of positively charged pharmacophore RWRWRW-NH2. A ruthenium-substituted deriva- and hydrophobic amino (16, 17). It is effective against Gram- tive of this localized to the membrane in vivo, and the positive including methicillin-resistant Staphylococcus peptide also integrated readily into mixed bilayers aureus strains, is moderately effective against Gram-negative bac- that resemble Gram-positive membranes. Proteome and Western teria, has low toxicity against human cell lines, and displays low blot analyses showed that integration of the peptide caused de- hemolytic activity (18). MP196 therefore is a promising lead localization of peripheral membrane proteins essential for respi- structure for derivatization and already has yielded peptides with

ration and cell-wall biosynthesis, limiting cellular energy and improved activities and altered pharmacological properties (19). MICROBIOLOGY undermining cell-wall integrity. This delocalization phenomenon For example, in a recent systematic L-to-D exchange scan peptides also was observed with the cyclic peptide gramicidin S, indicating with significantly reduced hemolytic activity could be identified (20). the generality of the mechanism. Exogenous glutamate increases Proteomic profiling of the Gram-positive bacterium tolerance to the peptide, indicating that osmotic destabilization subtilis exposed to MP196 provided a starting point for mechanistic also contributes to antibacterial efficacy. responds to peptide stress by releasing osmoprotective amino acids, in part Significance via mechanosensitive channels. This response is triggered by mem- brane-targeting bacteriolytic peptides of different structural clas- Multidrug-resistant bacteria present an acute problem to ses as well as by hypoosmotic conditions. medicine, generating interest in novel antimicrobial strategies. Antimicrobial peptides currently are being investigated, both as mechanism of action | respiratory chain | hypoosmotic stress response | and as immunomodulatory agents. Many antimicro- metallocenes bial peptides interact with the bacterial membrane, a previously underexplored target. We present a system-based study uring the past two decades, bacterial antibiotic resistance has of the mode of action of small cationic peptides and the mecha- Dgrown into a major threat to public health, restoring in- nisms that bacteria use to defend against them. We show that fectious diseases to the list of leading causes of death worldwide peptide integration into the membrane causes delocalization (www.who.int/healthinfo/statistics/bodgbddeathdalyestimates.xls). of essential peripheral membrane proteins. This delocalization As a reaction to this health crisis, several efforts currently are impacts on two cellular processes, namely respiration and cell- underway to discover and develop new natural and synthetic an- wall biosynthesis. We describe a bacterial survival strategy in tibiotic compounds. One promising antibiotic class is antimicrobial which mechanosensitive channels in the bacterial membrane peptides, which occur naturally as part of host defense systems in establish osmoprotection against membrane-targeting bacte- all domains of life (1–4). Antimicrobial peptides range in length riolytic peptides. Understanding the peptides’ mode of action from four to more than 100 amino acids and fall into a number of and bacterial survival strategies opens up new avenues for different structural classes, including α-helical , lipo- devising peptide-based antibacterial strategies. peptides, glycopeptides, lantibiotics, and short cationic peptides. The short cationic peptides offer a potent alternative to longer Author contributions: M.W., H.-G.S., and J.E.B. designed research; M.W., A.I.C., A.O., D.Z., natural antimicrobial peptides. The latter can be difficult to C.M., C.S., and R.G. performed research; H.B.A., M.P., U.K., N.M.-N., S.K.S., E.B., D.B., and H.B.-O. contributed new reagents/analytic tools; M.W., A.I.C., A.O., D.Z., C.M., C.S., isolate and are complicated to synthesize chemically, but short R.G., R.E., H.B.-O., and J.E.B. analyzed data; and M.W., H.B.-O., H.-G.S., and J.E.B. wrote cationic peptides are generated readily by solid-phase peptide the paper. synthesis and are easily accessible for chemical derivatization The authors declare no conflict of interest. (5–8).Theyarecharacterizedbypositivelychargedandby This article is a PNAS Direct Submission. M.Z. is a guest editor invited by the hydrophobic amino acids (9, 10). Previous mechanistic studies Editorial Board. in vitro have examined the interactions of the peptides with Data deposition: The mass spectrometry proteomics data have been deposited to the membranesormembraneextracts(11–15), but their effects on ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [Vizcaino JA, et al. (2013) The Proteomics Identifications (PRIDE) bacterial physiology have been largely underexplored. database and associated tools: Status in 2013. Nucleic Acids Res 41(D1):D1063–D1069]. A more complete understanding of how these short cationic The dataset ID is PXD000181. antimicrobial peptides bring about bacterial cell death is needed 1To whom correspondence should be addressed. E-mail: [email protected]. to further their optimization and development for practical This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. applications. To achieve this understanding, we studied the 1073/pnas.1319900111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1319900111 PNAS Early Edition | 1of10 Downloaded by guest on September 30, 2021 studies. It identified two major areas for analysis: membrane and S1–S4). Proteins indicative of general cell-envelope stress, mem- cell-wall integrity and energy metabolism. The proteome brane stress, energy limitation, and cell-wall stress were strongly analysis prompted further investigation into the deregulation of up-regulated upon exposure to MP196 (Fig. 1C), indicating that amino biosynthesis, which revealed that B. subtilis coun- the cell envelope is the primary target of MP196. From a library teracts the attack on the cell envelope by triggering an osmo- of proteome response profiles that includes the responses to protective release of glutamate. more than 50 antibiotic agents (22–26), several antibiotics were identified that share subsets of these marker proteins with Results MP196, all affecting the bacterial cell envelope (SI Appendix, Inhibition of Macromolecule Biosynthesis Points to the Bacterial Cell Table S5). The strongest overlap in up-regulated proteins was Envelope as a Target of MP196. To explore the inhibition of mac- observed for the membrane-targeting agents gramicidin S and romolecule biosynthesis by MP196 in vivo, we studied incorporation valinomycin. Gramicidin S, a member of the small cationic an- of radioactive precursors. Cells were exposed to MP196 concen- timicrobial peptide class, is structurally distinct from MP196: It trations that reduced growth rates by ∼50% unless noted otherwise is a cyclic amphipathic peptide that does not contain the ar- (SI Appendix,Fig.S2). MP196 moderately inhibited incorporation ginine or tryptophan residues found in MP196. It disrupts of DNA, RNA, protein, and cell-wall precursors (Fig. 1A and membrane function by integrating into the lipid bilayer (27). SI Appendix, Fig. S3). To separate the primary event from sec- Valinomycin is an uncharged dodecadepsipeptide, which acts as ondary effects, the activation of reporter genes was studied in a potassium-carrier ionophore. Both antibiotics were selected as B. subtilis (Fig. 1B). Each of the reporter strains had been shown close comparators to study the antibiotic mechanism of MP196. previously to respond specifically to the inhibition of a particular We also chose and as comparator compounds. cellular process using different antibiotics (21). MP196 did not They both share marker proteins of cell-envelope stress with activate transcription from promoters of yorB, helD,orbmrC, MP196. Vancomycin is a lipid II-binding cell-wall biosynthesis which are indicative of DNA damage, RNA synthesis inhibition, inhibitor, and nisin, in addition to inhibiting cell-wall bio- and translation inhibition, respectively. The liaI promoter, which synthesis by binding lipid II, forms heteromultimeric membrane is responsive to inhibition of steps in membrane-bound cell-wall pores with lipid II. biosynthesis, was activated weakly, whereas ypuA,whichisre- sponsive to both membrane-associated and membrane-independent A Stereospecific Binding Site Is Not Required for MP196 Target cell-wall stress, was strongly activated. Interaction. Given the proteomics results, it seemed likely that the lipid bilayer is a direct target of the peptide. However, to The Proteomic Response of B. subtilis Provides Additional Evidence exclude the possibility that MP196 binds to a protein target and That MP196 Targets the Cell Envelope. TheresponseofB. subtilis to downstream effects elicit the observed proteomic response, we treatment with MP196 was studied at the proteome level by investigated the importance of peptide stereochemistry for an- combining 2D gel-based and LC-MS–based approaches (SI Ap- tibacterial activity and MP196–target interaction using an all–D- pendix, Results of the Comparative Proteome Analysis and Tables peptide (D-MP196). D-MP196 was as active as MP196

Fig. 1. Narrowing down the target area. (A)[3H]-glucosamine incorporation by S. simulans upon treatment with MP196 or vancomycin. (B) Transcription activation of selected B. subtilis promoters indicative of inhibition of specific cellular processes. Cells were incubated with increasing of MP196 for predetermined times depending on the induction kinetics of the respective reporter strains. (C) 2D gel-based proteome analysis of MP196-treated B. subtilis. Synthesis rates of cytosolic proteins of MP196-treated (false-colored in red) and untreated (false-colored in green) B. subtilis were compared based on [35S]-methionine labeling. In the overlaid autoradiographs, down-regulated proteins appear green, up-regulated proteins appear red, and proteins expressed at equal rates appear yellow. Unidentified proteins are marked by circles. Blue labels indicate marker proteins for general cell-envelope stress, green labels identify cell-wall biosynthesis inhibition, and red labels are markers for membrane stress.

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1319900111 Wenzel et al. Downloaded by guest on September 30, 2021 against B. subtilis (18), and the two forms had highly similar PNAS PLUS proteome responses (SI Appendix,Fig.S4A and B). Thus, stereochemistry is not crucial for target interaction, making it unlikely that MP196 binds to a specific site on a protein target.

MP196 Affects Cell Morphology. The impact of nonlytic concen- trations of MP196 and gramicidin S on the morphology of B. subtilis cells was examined by transmission electron microscopy (TEM) (Fig. 2A). MP196-treated cells displayed characteristic cell-wall lesions. The at these sites is thinner, suggesting a loss of integrity. Gramicidin S-treated cells did not retain their shape during sample preparation, culminating in the leakage of cell contents and cell collapse. Thus, although both peptides in- duce similar proteome-response patterns, the structural impair- ment of the B. subtilis cell envelope is more severe after gramicidin S treatment than after MP196 exposure.

MP196 Accumulates in the Bacterial Cell Envelope. To study the subcellular localization of MP196 in vivo, a ruthenocene- substituted derivative Rc-C(O)-WRWRW-NH2 (MP276) (see SI Appendix,Fig.S1for structure) was synthesized for TEM and atomic absorption spectrometry studies. The ruthenocene sub- stitution did not affect antibacterial activity negatively (18), and ruthenium alone is not toxic. Thin sections of untreated and MP276-treated B. subtilis were prepared omitting the lead-staining step, which is used as con- trast agent in electron microscopy (Fig. 2B). Accordingly, in the

preparations of untreated cells the cell envelope is poorly con- MICROBIOLOGY trasted compared with the cytosol. However, in MP276-treated cells the cell envelope is seen in high contrast, showing that the peptide with the electron-dense ruthenium label accumulates in the cell envelope.

Peptide Concentrations Are Highest in the Bacterial Membrane. Graphite furnace atomic absorption spectrometry provides ab- solute quantitation of the ruthenium-labeled peptide in subcellular fractions. Because ruthenium does not occur naturally in bacteria, it is ideally suited for peptide tracing in the cellular environment (28). The absolute ruthenium amounts were related to the volumes of the cellular compartments to give compartment concentrations (SI Appendix,TableS6). Volumes were calculated based on high- resolution cryo-electron microscopy studies of the B. subtilis membrane and cell-wall structures (29, 30). The ruthenium con- centration in the membrane fraction exceeded concentrations measured in the cytosolic and cell-wall fractions by factors of 15 and 17, respectively. In agreement with peptide tracing by TEM, the principal localization of MP276 is the membrane. During sample preparation, cells were washed with EDTA several times. − There was no significant elution of ruthenium (∼10 21 mol per cell), suggesting strong binding of the peptide to the phospholipid Fig. 2. Peptide effects on cells and subcellular localization. (A) TEM images bilayer. Lower but still significant amounts of peptide were of peptide-treated B. subtilis. Cells were fixed with 2% uranyl acetate, and ultrathin sections were stained with 0.2% lead citrate in 0.1 M NaOH. (B)In detected in the cell wall, which can function as a barrier that vivo peptide tracing using the ruthenocene-substituted MP196 derivative detains antibiotics (31). Concentrations of ruthenium above back- MP276. B. subtilis cells were fixed with 2% uranyl acetate. Sections remained ground levels also were detected in the cytosolic fraction, sug- unstained to avoid interference of lead with the ruthenium signal. Ruthe- gesting that the peptide has some ability to cross the lipid bilayer. nium in the cytosol, membrane, and cell wall was quantified by atomic ab- sorption spectroscopy. MP196 Integrates into Phospholipid Bilayers That Mimic Gram- Positive Membranes but Not Erythrocyte Membranes. Interaction of MP196 with the membrane was investigated by differential the thermograms indicates that MP196 integrates into the phos- scanning calorimetry (DSC) using model membranes consisting pholipid bilayer, generating “peptide-affected domains” (14). of the two most abundant in the B. subtilis These are characterized by a broadening of the existing transition membrane (32), namely 1,2-dipalmitoyl-sn-glycero-3-phospha- and the appearance of a small transition at lower temperatures, tidylglycerol (DPPG) and 1,2-dipalmitoyl-sn-glycero-3-phos- reflecting rearrangement of the lipid mixture, and, consequently, phatidylethanolamine (DPPE), in an 88:12 ratio (Fig. 3A). DSC alterations in packaging of the lipids at the emerging borderlines. monitors the thermotropic-phase behavior of the lipid bilayer. When bilayers composed of a single type of phospholipid were Changes in pattern are indicative of perturbation of the mem- used, the influence of MP196 was far less pronounced. A brane bilayer and packaging of the phospholipids’ fatty acyl broadening of the shoulder at lower temperatures indicated the chains and, therefore, of peptide integration. Deconvolution of formation of some peptide-affected domains on DPPG bilayers,

Wenzel et al. PNAS Early Edition | 3of10 Downloaded by guest on September 30, 2021 MP196 Does Not Disturb Metal Cation Homeostasis. The B. subtilis proteomic responses to MP196 and to the potassium ionophore valinomycin were markedly similar. To investigate whether MP196 integration into the membrane disturbs metal cation homeostasis, total cellular concentrations were determined after peptide treatment in chemically defined culture broth (SI Appendix, Fig. S6B). In agreement with the observed lack of potassium efflux in choline buffer, no significant decrease in any metal ion was observed after MP196 treatment. In contrast, gramicidin S treatment caused a significant decrease in potas- sium, magnesium, and manganese levels. Valinomycin-treated cells selectively accumulated potassium (an eightfold increase), probably because of overcompensation. These results fit well with the observation that, despite provoking a strong proteome response, valinomycin does not inhibit B. subtilis growth (24).

MP196 Integration Causes Membrane Depolarization. Many mem- brane-targeting antibiotics, including gramicidin S and valinomy- cin, depolarize the bacterial membrane (27, 34). Depolarization was investigated using a B. subtilis strain carrying a GFP fusion to the cell division-regulating protein MinD. Normally localized at the cell poles and in the cell-division plane, MinD delocalizes upon depolarization, resulting in a spotty pattern of GFP-MinD distribution (35). In contrast to the normal MinD pattern in the untreated control cells, MinD delocalization was seen in MP196 and gramicidin S-treated cells (Fig. 4A). Depolarization upon MP196 treatment was confirmed in B. megaterium with the volt- age-sensitive fluorescent probe DiSC35(SI Appendix,Fig.S6C).

Fig. 3. Interaction with the membrane. (A) DSC thermograms of lipid ATP Levels Drop in MP196-Treated Cells. Sudden depolarization bilayers consisting of 88:12 DPPG/DPPE incubated with MP196. Changes in should result in energy limitation, and energy limitation also thermotropic-phase behavior caused by perturbation of fatty acyl packing would be consistent with the proteome response. Intracellular indicate peptide integration. (B) DSC thermograms of lipid bilayers con- sisting of DPPC. (C) Overlaid fluorescence microscopy images of Bac- ATP levels of B. subtilis, determined using a luciferase assay, Light-stained B. subtilis treated with valinomycin, nisin, gramicidin S, and showed a 60% reduction in cellular ATP content in MP196- MP196. A red-fluorescing dye selectively stains cells with large membrane treated cells and a 70% reduction in gramicidin S-treated cells holes; a green-fluorescing dye stains all cells independently of membrane (Fig. 4B), whereas valinomycin and nisin caused 30% drops. integrity. Cells with intact membranes appear green, and cells with perfo- Thus, MP196 has a considerable impact on energy metabolism. rated membranes appear orange. No significant ion efflux was observed under the test conditions, indicating that depolarization and energy limitation are not caused solely by ion transfer. but no effect was seen using pure DPPE (SI Appendix, Fig. S5)or pure 1,2-dipalmitoyl-sn-glycero-3- (DPPC) MP196 Inhibits the Respiratory Chain at the Cytochrome c Level. (Fig. 3B), which typically are used to model erythrocyte mem- Because of the lack of pore formation and ion transfer, which branes (33). That MP196 affected DPPG but not DPPE implies are typical causes of a breakdown of energy metabolism, we tested a preference for negatively charged over neutral lipids and could whether components of the respiratory chain are inhibited by explain the preference for bacterial over erythrocyte membranes. MP196. Inhibition of ATP synthase would limit ATP directly. This supposition is consistent with the known low hemolytic ac- Proton-pumping activity was monitored in Micrococcus flavus tivity of MP196 (18). A preference for negatively charged phos- inverted vesicles using the pH-sensitive probe acridine orange pholipids has been shown previously for other very small RW- (SI Appendix,Fig.S6D). The ATP synthase inhibitor lacto- rich peptides, Combi-1 (Ac-RRWWRF-NH2) and LfcinB4–9 ferrin (36) served as a positive control. No inhibition of ATP (RRWQWR-NH2), using the same method (reviewed in ref. 13). synthase activity was observed at MP196 concentrations cor- responding to those used in the proteome analysis. Peptide Integration Does Not Lead to Pore Formation. We used Inhibition of the respiratory chain upstream of ATP synthase fluorescence staining to test whether in vivo integration of the would contribute to the breakdown of the membrane potential, MP196 peptide results in pore formation in B. subtilis. The green- resulting in the loss of ATP synthesis. Using M. flavus inverted fluorescent dye SYTO 9 crosses intact membranes, whereas the vesicles, inhibition of the respiratory chain was monitored with red-fluorescent propidium iodide enters bacterial cells through iodonitrotetrazolium chloride, a reduction-sensitive dye (Fig. lesions in the membrane (Fig. 3C). Pore-forming nisin allowed 4C). Rotenone and antimycin A, specific inhibitors of complexes both dyes to enter B. subtilis cells, resulting in orange cells. MP196 I and III of the respiratory chain, respectively, were used as treatment, as well as treatment with non–pore-forming gramicidin comparator compounds. Rotenone reduced electron transport S and valinomycin, resulted in green-fluorescent cells—like the activity by 20%, and antimycin A did so by 50%. MP196 dis- untreated control—suggesting that MP196 does not act by form- played similar inhibition efficiency, reducing electron transport ing large pores. The same conclusions were drawn from measuring by 30%. MP196 inhibition was additive with either rotenone or peptide-induced potassium leakage from antimycin A, suggesting that MP196 inhibits a different compo- cells into choline buffer (SI Appendix,Fig.S6A). In contrast to nent of the respiratory chain. the positive control, nisin, MP196 released little potassium, even Cytochrome c, which is located on the outer membrane leaflet, at concentrations equivalent to 50 times the minimal inhibitory transfers electrons from complex III to complex IV. Its locali- (MIC). zation after MP196 treatment was investigated by Western blot

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1319900111 Wenzel et al. Downloaded by guest on September 30, 2021 PNAS PLUS MICROBIOLOGY

Fig. 4. Effects on membrane potential and respiration. (A) Fluorescence microscopy images (Upper Row) and corresponding bright-field images (Lower Row) show GFP-MinD localization in untreated B. subtilis cells and cells incubated with gramicidin S or MP196. GFP-MinD delocalization indicates membrane depolarization. (B) ATP levels of B. subtilis cells treated with valinomycin, nisin, gramicidin S, or MP196. In a luciferase assay, ATP concentrations were de- termined under conditions resembling those of the proteome experiment. (C) Activity of the respiratory chain of inverted M. flavus vesicles treated with rotenone, antimycin A, or MP196. Electron transport efficiency was monitored by the reduction of iodonitrotetrazolium chloride causing a decrease in absorbance at 485 nm. (D) Western analysis of cytochrome c in membrane fractions of B. subtilis after a 5-min treatment with gramicidin S and MP196. The Ponceau S-stained blot is displayed as loading control. (E) Overview of the bacterial respiratory chain [according to Vonck and Schäfer (38)]. Proton move- ments are indicated by green arrows; electron movements are indicated by orange arrows.

analysis of membrane fractions of antibiotic-treated B. subtilis cells as did gramicidin S (although gramicidin S is not known to in- (Fig. 4D). The amount of cytochrome c in the membrane was terfere directly with cell-wall biosynthesis). reduced drastically after 5 min of treatment with MP196, in- A stereospecific target interaction was excluded by experi- dicating dissociation of the protein from the extracellular mem- ments with the D-MP196 variant. Therefore we investigated brane surface. This delocalization is independent of the membrane whether inhibition of a membrane-bound step of cell-wall bio- potential breaking down, because cytochrome c was detected in synthesis could be attributed to the delocalization of a peripheral the membrane fraction of the untreated control cells, which were , similar to the inhibition of the respiratory disrupted by ultrasonication. Rather, delocalization requires in- chain by cytochrome c delocalization. In B. subtilis, the only teraction of the peptide with the membrane. A similar effect was peripheral membrane protein in the pathway is MurG, the en- observed using gramicidin S, which previously was shown to inhibit zyme that converts lipid I to lipid II by attaching GlcNAc to the components of the respiratory chain (37). Detachment of cyto- carrier-conjugated UDP-N-acetylmuramic acid chrome c from the outer membrane leaflet explains the inhibition (UDP-MurNAc) pentapeptide . When the localization of the respiratory chain, breakdown of the membrane potential, of MurG was investigated by Western blot analysis (Fig. 5B), and subsequent energy limitation (Fig. 4E) (38). both MP196 and gramicidin S treatment caused almost complete loss of MurG from the membrane fraction of treated B. subtilis MP196 Undermines Cell-Wall Integrity by Delocalizing Another within 5 min. The removal of MurG from the cytosolic mem- Essential Peripheral Membrane Protein. Following MP196 expo- brane surface, in combination with energy limitation (see above), sure, cell-wall biosynthesis is slightly inhibited (as revealed by may explain the reduced glucosamine incorporation, loss of cell- precursor incorporation), severe cell-wall lesions form (as revealed wall integrity, and induction of a cell-wall–specific stress response. by TEM), and there is a significant overlap of marker proteins Inhibition of earlier cytosolic steps of cell-wall biosynthesis, in- with compounds that target membrane-bound steps of cell-wall hibition of membrane-bound steps by lipid II binding, and direct biosynthesis in proteomic studies. When incorporation of cell-wall inhibition of MurG activity were excluded experimentally (SI material is inhibited at a step in membrane-bound biosynthesis, Appendix, Results of Cell Wall Biosynthesis Inhibition Assays B. subtilis cells undergo a characteristic change in shape after and Fig. S7). acetic acid/methanol fixation, namely extrusion of the cell mem- brane through holes in the cell wall (4, 26). Untreated cells and MP196 Integration Triggers MurG Release from Isolated B. subtilis cells treated with valinomycin did not display membrane extru- Membranes. Western analysis of disrupted cell membranes revealed sions (Fig. 5A); nisin and MP196 did induce membrane extrusions, that both cytochrome c and MurG bind the membrane independent

Wenzel et al. PNAS Early Edition | 5of10 Downloaded by guest on September 30, 2021 downshift and treatment with bacteriolytic peptides that target the membrane: the cyclic and cationic gramicidin S, the α-helical cat- ionic aurein 2.2, the cationic lantibiotic nisin, or the uncharged amphiphilic gramicidin A. It was not observed after osmotic upshift or treatment with valinomycin or vancomycin, peptides that are either not bacteriolytic or do not target the membrane (Fig. 6F). Mechanosensitive channels appear to contribute to glutamate release after MP196 treatment. A B. subtilis mutant that lacks the four known MscL and MscS-type mechanosensitive channels (39) accumulated high levels of glutamate intracellularly (Fig. 6G) and showed heightened sensitivity to MP196 (Fig. 6 C–E). However, these channels are not the only route for glutamate release, because extracellular glutamate levels increased when the quadruple msc mutant was treated with MP196 (Fig. 6B). Discussion The cationic hexapeptide MP196, which is the minimal phar- macophore of RW-rich peptides, was used here to study the mechanism of action of short cationic antimicrobial peptides and subsequent bacterial adaptation strategies. MP196 acts on the bacterial cytoplasmic membrane (SI Appendix, Fig. S9 A, I), Fig. 5. Effects on the cell wall. (A) Light microscopy images showing cell-wall where essential physiological processes, including respiration (SI integrity of B. subtilis after treatment with MP196 and nisin. Acetic acid- Appendix, Fig. S9 A, II), cell-wall biosynthesis (SI Appendix, Fig. methanol fixation visualizes cell-wall damage by extrusions of the membrane S9 A, III), and membrane transport SI Appendix, Fig. S9 A, IV), through holes in the cell wall. (B) Western blot detection of the cell-wall biosynthesis protein MurG in membrane fractions isolated from B. subtilis take place. At the molecular level (SI Appendix,Fig.S9B, I), cells that were treated with peptide for 5 min. (C) Western blot detection of MP196 integrates into the lipid bilayer readily and disturbs MurG in B. subtilis membrane fractions that first were isolated and then were membrane architecture, as revealed by the perturbation of fatty incubated with peptide for 5 min. acyl packing in the DSC analysis. This membrane integration likely is promoted by the interaction of the cationic arginine residues with negatively charged phospholipid head group and is facilitated of the membrane potential. To test if MP196 integration is sufficient by lipophilic tryptophan residues. Such a mechanism of membrane for the release of MurG from the membrane, the membrane frac- integration is typical of amphipathic peptides (13, 40–42), which tion of disrupted B. subtilis was collected and incubated with the accumulate in the membrane near the interface with the cytosol, peptide in vitro (Fig. 5C). Western blot analysis showed that MurG making contact with lipid head groups and fatty acyl chains. was released efficiently from the MP196-treated membrane fraction RW-rich peptides have been shown to differ in their ability to but remained attached to the untreated membrane, demonstrating permeabilize membranes. Although the tridecapeptides tritrpti- that MP196 is sufficient for MurG detachment from the membrane. cin and indolicidin permeabilize lipid bilayers (43), hexapeptides such as Combi-1 and Combi-2 showed very poor permeabiliza- B subtilis . Counteracts MP196 Action by Osmotic Stabilization. Pro- tion (44). It is important to note that even peptides with very teome analysis revealed up-regulation of proteins involved in small structural differences can have distinct mechanisms of amino acid anabolism, especially of glutamate and aspartate, interacting with membranes, resulting in different permeabiliza- within 65 min of MP196 treatment (SI Appendix, Table S3). To tion capabilities and modes of action, as shown recently for very investigate the influence of MP196 on the amino acid pool, in- small cyclic RW-rich peptides (45). Thus, the observations tracellular and extracellular free amino acid levels were mea- noted in this study may not necessarily apply to all antimicro- sured by HPLC after 15 min of antibiotic treatment. MP196- bial peptides. treated cells accumulated free arginine and intracellularly For MP196 we found no evidence for membrane permeabiliza- (Fig. 6A and SI Appendix, Fig. S8A), consistent with the up- tion or pore formation in model membranes or in vivo. No ion regulation of proteins involved in valine biosynthesis (SI Ap- release occurs in chemically defined bacterial culture medium. It pendix, Table S3). Intracellular glutamine/glutamate, asparagine/ has been suggested that integration of cationic amphiphatic pep- aspartate (the assay used here does not distinguish between tides leads to local lipid reorganization and the formation of glutamate and glutamine or between aspartate and asparagine), “peptide-induced lipid pores” that allow metal to cross the , and proline levels were substantially reduced, with con- membrane (42); however, the lack of ion release in chemically de- comitant dramatic increases in extracellular glutamine/glutamate fined medium suggests that ion efflux is not a major effect of MP196 and asparagine/aspartate and moderate increases in extracel- under the growth conditions used in the proteome experiment. lular arginine, lysine, and proline (Fig. 6B and SI Appendix, The integration of MP196 into the membrane affects multiple Fig. S8B). The absolute amounts of glutamine/glutamate re- cellular processes (SI Appendix, Fig. S9 B, II–IV). Like the RW- leased exceeded by far the sum of intracellular and extracellular rich small cationic antimicrobial peptide Bac8c (46) and the levels of untreated B. subtilis cultures, indicating dedicated bio- small cyclic cationic peptide gramicidin S (37) that does not synthesis and export (SI Appendix,Fig.S8C). contain arginine or tryptophan residues, MP196 has a profound Supplementing defined medium with exogenous glutamate impact on the bacterial respiratory chain (SI Appendix, Fig. S9 B, increased the MIC of MP196 against B. subtilis by up to eightfold II). Our data suggest that the inhibition of the respiratory chain (Fig. 6C), establishing that glutamate has a protective effect by small cationic antimicrobial peptides is caused by the de- against MP196-induced stress. Similar results were obtained by tachment of cytochrome c from the membrane, resulting in supplementation with exogenous sodium chloride (Fig. 6D) and disruption of the electron transfer chain and subsequent break- potassium chloride (Fig. 6E), suggesting that the protection against down of the proton gradient. Inhibition of the respiratory chain MP196isnotspecificforglutamatebutiscausedbyosmoticeffects. then affects ATP synthesis, causing energy limitation that Glutamate release also was observed in response to osmotic impacts all other macromolecule biosynthesis pathways.

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1319900111 Wenzel et al. Downloaded by guest on September 30, 2021 PNAS PLUS

Fig. 6. Amino acid composition. (A) HPLC analysis of the intracellular amino acid composition of B. subtilis treated with MP196. (B) Extracellular amino acid MICROBIOLOGY composition of the same cultures. Only the amino acids whose concentrations changed significantly after peptide treatment are displayed here (see SI Appendix, Fig. S8 for full amino acid profiles). Amino acids are written in a one-letter code in the order of elution time from the column. Glutamate and glutamine as well as aspartate and asparagine are not distinguishable by this method and appear as one peak each. Tryptophan was not quantified here. (C– E) MICs of MP196 against B. subtilis 168, a B. subtilis strain lacking all known mechanosensitive channels (SMB80), and its parent strain (JH642) in defined medium supplemented with increasing glutamate (C), NaCl (D), or KCl (E) concentrations. MICs were determined independently twice. (F) Intra- and ex- tracellular glutamate concentrations in B. subtilis cells under different antibiotic and osmotic stress conditions determined by amino acid analysis. (G) Intra- and extracellular glutamate concentrations in a B. subtilis JH642 and SMB80.

The impact of MP196 on cell-wall biosynthesis is illustrated by helix motif (35, 52), whereas cytochrome c variants in B. subtilis a reduction in precursor incorporation, promoter activation of attach to the outer leaflet of the membrane by a lipid anchor cell-wall stress-responsive genes, up-regulation of proteins in- or transmembrane helices (53, 54). Thus, MP196 causes de- dicative of cell-wall biosynthesis stress, and loss of cell-wall in- localization of proteins on both sides of the membrane, which tegrity. Similar observations have been reported for gramicidin S attach to the membrane by different mechanisms. It is possible (26) and Bac8c (46). In addition to an indirect effect on cell-wall that, in addition to the proteins studied here, other membrane- biosynthesis caused by energy limitation, we found that small associated proteins are delocalized as a consequence of MP196- cationic peptides affect cell-wall biosynthesis by delocalizing the mediated membrane injury. lipid II biosynthesis protein MurG from the intracellular surface Lacking evidence of a stereospecific binding site for MP196, no of the membrane. As with cytochrome c, protein delocalization is specific receptor–ligand interaction is apparent from our studies. independent of the membrane potential. Rather, it seems to be In fact, our data suggest that the molecular target of MP196 is the a consequence of the alterations in membrane architecture lipid bilayer itself, and no other specific target than the membrane caused by peptide integration, which were observed by DSC. is identified. However, the main functional impact of MP196 is on Delocalization of MurG could explain the effects on cell-wall proteins located in the membrane. Therefore MP196 interferes biosynthesis and integrity exerted by MP196. This enzyme re- simultaneously with a broad range of cellular processes, compli- cently was shown to be the direct target of the anti-staphylo- cating the development of bacterial resistance. DSC experiments coccal small molecule inhibitor murgocil (47). showed that the interaction of MP196 with the membrane criti- We suggest that delocalization of peripheral membrane pro- cally depends on lipid composition, thereby offering the oppor- teins is a general mechanism extending to membrane-targeting tunity to design peptides selective for Gram-positive and Gram- peptides of other structural classes such as the negative organisms over mammalian cells. . Daptomycin has been shown to alter membrane The proteome response and amino acid analysis provide evi- architecture and to delocalize the cell-division protein DivIVC, dence that B. subtilis counteracts peptide-mediated membrane which binds the membrane in a curvature-dependent fashion stress by inducing three known defense strategies: adjustment of (48–50). Although delocalization of MurG by daptomycin has membrane lipid composition (55–57), stabilization of the mem- not been described thus far, it would explain the induction of the brane (58, 59), and restriction of access to the membrane by the cell-wall stress response (51). enhancement of wall teichoic acid D-alanylation (SI Appendix, A further consequence of MP196 treatment is the de- Fig. S9 C, I–III; see SI Appendix for details) (31, 60–62). Here we localization of the cell-division regulating protein, MinD. MinD describe another survival strategy, one that entails the release of previously was shown to delocalize upon membrane depolarization selected amino acids from the cells (SI Appendix, Fig. S9 C, IV). (35). MinD and MurG attach to the intracellular surface of the Large quantities of glutamine/glutamate, plus significant amounts membrane through electrostatic interactions using an amphipathic of asparagine/aspartate, arginine, proline, and lysine, were released

Wenzel et al. PNAS Early Edition | 7of10 Downloaded by guest on September 30, 2021 into the medium, and biosynthesis of these amino acids was Radioactive Precursor Incorporation. The influence of MP196 on the major reflected in the proteome response. macromolecular synthesis routes was studied by incorporation of radioac- The release of amino acids is mediated in part by mechano- tively labeled precursor by Staphylococcus simulans as described sensitive channels, which measure membrane tension, and their by Schneider et al. (76). transient opening is triggered by turgor-induced pressure on the lipid bilayer (63). In plants, the activation of host defense by Reporter Gene Activation. Damage to the main cellular macromolecules was monitored by promoter activation of selected marker genes fused to the bacterial antimicrobial peptides has been attributed to mecha- firefly luciferase reporter gene in the genetic background of B. subtilis IS34. noreceptors directly sensing peptide-induced alterations in mem- – μ – Serial twofold dilutions of each peptide (0.031 64 g/mL) were inoculated brane architecture (64 66). with bacterial cell suspensions and incubated at 37 °C for a time period Glutamate is an osmoprotectant that accumulates intracellularly depending on the induction kinetics of the reporter strain. Then 2 mM lu- in some bacilli. In B. subtilis it is the most abundant free amino acid, ciferin was added, and flash luminescence was measured. with a pool size of about 100 mM (67). Under hyperosmotic stress the intracellular glutamate pool Proteomics. Treatment of B. subtilis 168, metabolic labeling of newly syn- 35 increases moderately (up to about 170 mM) (67). Under these thesized proteins with [ S]-L-methionine, and subsequent protein separa- conditions the outflow of is prevented by increasing pro- tion by 2D PAGE were performed as described previously (25). For gel-free line levels (depending on the severity of the environmentally proteome analysis of membrane proteins, B. subtilis 168 was grown aero- bically at 37 °C in Belitzky minimal medium (BMM) supplemented with imposed osmotic stress) up to pool sizes of 500 mM in severely 14 14 stressed cells (68–70). Upon osmotic downshock the opposite is N-ammonium sulfate and N-L-tryptophan. Cells were stressed at an OD500 of 0.4 with 22.5 μg/mL MP196 for 65 min or were left untreated as control. the case: releases potassium glutamate into the 15 15 Untreated cultures grown on N-ammonium sulfate and N-L-tryptophan medium using mechanosensitive channels (71). In Corynebacterium were used for relative quantification. Mixing of cell extracts before subcellular glutamicum glutamate excretion through mechanosensitive channels fractionation steps for relative quantification was carried out according to is triggered in response to penicillin treatment (72). We show here Otto et al. (77). The enriched membrane protein fraction was prepared that B. subtilis also releases glutamate in response to hypoosmotic according to the workflow published by Eymann et al. (78), omitting the stress conditions. Glutamate release is triggered further in response n-dodecyl-β-D-maltoside treatment step. The preparation of the integral to treatment with membrane-targeting bacteriolytic peptides be- membrane peptides was carried out as described by Wolff et al. (79). Sample longing to different structural classes (cyclodecapeptide gramicidin preparation, mass spectrometric measurement, and subsequent data analysis S, α-helical aurein 2.2, lantibiotic nisin, and uncharged were carried out as described by Otto et al. (77). The mass spectrometry pro- amphiphilic gramicidin A) (Fig. 6F). These results suggest that teomics data have been deposited with the ProteomeXchange Consortium osmoprotective glutamate releaseisageneralreactiontomem- (http://proteomecentral.proteomexchange.org) via the PRIDE partner brane-targeting bacteriolytic peptides. The addition of exogenous repository (80) with the dataset identifier PXD000181. glutamate to the growth medium protected against MP196 via os- motic stabilization (Fig. 6 C–E). Similar observations were made for TEM. Cells were grown in BMM to an OD500 of 0.35. The main culture then was subdivided into 50-mL aliquots, and subcultures were treated with the sublancin 168, a glycopeptide with an unknown mechanism of ac- respective antibiotics for 15 min or were left untreated as control. Cells were tion produced by B. subtilis 168. High NaCl concentrations lowered harvested by centrifugation and washed twice in 100 mM Tris/1 mM EDTA, the susceptibility of sublancin-sensitive strains (73). High salt con- pH 7.5, and subsequently were washed once in the same buffer without centrations also negatively affected the susceptibility of S. aureus EDTA. Bacterial samples for TEM were prepared according to Santhana Raj strains to the human antimicrobial peptides LL-37 (α-helical) and et al. (81) with some modifications (see SI Appendix, Experimental Details). HNP-1 (β-sheet ) (74) as well as to the bactericidal activity To study effects on the cell envelope, sections were stained with 0.2% lead of the small cationic peptide thrombin-induced platelet microbicidal citrate in 0.1 M NaOH for 3 s. Samples were examined at 23,000–230,000× protein tPMP (75). magnification with a Philips EM410 transmission electron microscope This report provides a detailed physiological study of the in equipped with a Gatan digital camera system at an accelerating voltage vivo mechanism of action of a short RW-rich antimicrobial pep- of 80 kV. tide, which we chose as a model to study the action of short For peptide tracing, cell cultures were treated with 1 μg/mL of the ruth- cationic antimicrobial peptides. It illustrates how MP196 inte- enocene-substituted MP196 derivative MP276 (SI Appendix, Fig. S3D)for grates into the membrane, leading to delocalization of peripheral 15 min before sample preparation for TEM. The lead-staining step was omitted. membrane proteins. This delocalization includes the detachment Graphite Furnace Atomic Absorption Spectrometry. Cytosolic, membrane, and of the essential proteins cytochrome c and MurG from the cy- cell-wall fractions of untreated and MP276-treated B. subtilis were prepared. toplasmic membrane, impacting energy metabolism and cell-wall To this end, cells were harvested and washed five times with 100 mM Tris/1 biosynthesis. We propose that substantial energy limitation and mM EDTA before ultrasonication, centrifugation, and ultracentrifugation. loss of cell-wall integrity resulting from the delocalization of Ruthenium contents were quantified using a Vario 6 graphite furnace atomic essential peripheral membrane proteins are the major factors absorption spectrometer (Analytik Jena) as described previously (82–84). that contribute to MP196-mediated bacterial cell death. These perspectives on the action of small cationic antimicrobial pep- Differential Scanning Calorimetry. Membrane integration was investigated by tides, involving lipids and proteins, are complementary to the DSC-based determination of peptide-induced changes in the thermotropic- lipid-focused pore formation and carpet mechanism models. phase behavior of liposomes consisting of pure DPPG, pure DPPE, pure DPPC, Based on the data presented here, we suggest that MP196 action and a mixture of 88% DPPG and 12% DPPE, respectively. follows the interfacial activity model described by Wimley and Hristova (11), whereby, at a reduction in growth rate of Permeability Assays. In vivo pore formation in B. subtilis 168 was monitored 50%, phospholipid perturbation predominates over membrane using the Live/Dead BacLight bacterial viability kit (Invitrogen) following the ’ disruption. manufacturer s instructions. Potassium release from B. megaterium was performed as described previously (26). We also describe a bacterial defense strategy against bacteri- olytic membrane-targeting peptides that relies on lowering the Global Ion Analysis. Ionomics experiments were performed with B. subtilis 168 considerable osmotic potential of the B. subtilis cell, and hence in minimal medium under the same conditions as the proteomics experi- the magnitude of turgor (67), through the release of selected ments. Ion concentrations were determined by inductively coupled plasma amino acids into the medium. atomic emission spectroscopy.

Materials and Methods Membrane Potential Measurements. GFP-MinD localization assays were per- Experimental details and citations for all methods are provided in SI Appendix. formed with B. subtilis 1981 GFP-MinD (35) as described previously (26).

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1319900111 Wenzel et al. Downloaded by guest on September 30, 2021 PNAS PLUS Determination of membrane potential changes by use of DiSC35 was per- applied onto TLC plates (TLC Silica Gel 60 F254; Merck). Chromatography was formed as described by Andrés and Fierro (36) in B. megaterium. performed in chloroform-methanol-water-ammonia (88:48:10:1) and stained with phosphomolybdic acid stain at 140 °C. Accumulation of UPP-MurNac ATP Assay. ATP concentrations were determined from B. subtilis cytosolic pentapeptide was analyzed by HPLC as described by Schmitt et al. (88). extracts using the Perkin-Elmer ATPlite 1 step assay according to the manufacturer’s instructions. Amino Acid Analysis. Amino acid analysis of B. subtilis 168 (trpC2), B. subtilis JH642 (trpC2 pheA1), and SMB80 (trpC2 pheA1 mscL yhdY ykuT yfkC) (41) ATPase and Respiratory Chain Assays. M. flavus inverted vesicles for + cellular extracts and culture supernatants was performed by HPLC using the H -ATPase and respiratory chain activity measurements were prepared Acquity HPLC and AccQ•Tag Ultra system ( GmbH) according to the according to Burstein et al. (85). Proton pumping into M. flavus inverted ’ + manufacturer s instructions. Before HPLC, proteins were removed by vesicles by H -ATPase was measured in a microtiter plate assay based on acetone precipitation. the pH-sensitive probe acridine orange as described by Palmgren et al. MIC values against B. subtilis strains were determined with 5 × 105 cells/mL (86). Antibiotic influence on the bacterial electron transport chain was monitored by reduction of iodonitrotetrazolium chloride using M. flavus in BMM supplemented with increasing concentrations of glutamate, sodium inverted vesicles as described by Smith and McFeters (87). Cytochrome chloride, or potassium chloride. The lowest concentration inhibiting visible c localization was determined by Western blot detection of cytochrome growth was taken as the MIC. c on the same blots used for MurG detection. ACKNOWLEDGMENTS. We thank Michaele Josten, Petra Düchting, Monika Cell-Wall Integrity and Inhibition of Biosynthesis. Sample preparation and Bürger and Beate Menzel, and Stephanie Tautges, Kathrin Barlog, and Jale Stoutjesdijk for excellent technical assistance; Christoph H. R. Senges for help bright field microscopic inspection of B. subtilis 168 was performed as de- in preparing B. subtilis samples; Sina Langklotz and Dirk Albrecht for assis- scribed previously (26). tance with mass spectrometry; Helmut Meyer for providing amino acid anal- To detect MurG in membrane fractions of B. subtilis 168, cells were grown ysis technology; Klaus Funke and Ellen Kloosterboer for providing the in BMM until early logarithmic growth phase, treated with antibiotics for 5 cytochrome c antibody; Tanneke de Blaauwen and Franz Naberhaus for pro- min, and harvested by centrifugation. Membrane fractions were separated viding the MurG antibody; and AiCuris, GmbH for providing the B. subtilis by differential centrifugation, subjected to SDS PAGE, and blotted onto a reporter strains. This manuscript was written in part at the 2013 Scientific nitrocellulose membrane. MurG was detected with an E. coli MurG-specific Writing workshop of the Ruhr University Bochum (RUB) Research School; we antibody produced in rabbit and a secondary goat anti-rabbit IgG-HRP thank Lars Leichert and Richard Gallagher for critically reading the manuscript and for many helpful suggestions. This work was supported by a grant from conjugate (BioRad). the German federal state of North Rhine-Westphalia and the European Inhibition of in vitro lipid II synthesis was performed using M. flavus DSM Union (European Regional Development Fund “Investing in your future”) (to 1790 membrane preparations as described by Schneider et al. (76). For in N.M.-N., H.B.-O., H.-G.S., and J.E.B.) and by the RUB Research Department vitro lipid II binding, peptides were incubated with 2 nmol lipid II in 1:1 Interfacial Systems Chemistry (N.M.-N. and J.E.B.). C.M. was supported by the MICROBIOLOGY molar ratios. Reaction mixtures were incubated at 30 °C for 30 min and Protein Unit for Research in Europe, a project of North Rhine-Westphalia.

1. Giuliani A, Pirri G, Nicoletto SF (2007) Antimicrobial peptides: An overview of 18. Albada HB, et al. (2012) Modulating the activity of short arginine-tryptophan con- a promising class of therapeutics. Cent Eur J Biol 2(1):1–33. taining antibacterial peptides with N-terminal metallocenoyl groups. Beilstein J Org 2. Riedl S, Zweytick D, Lohner K (2011) Membrane-active host defense peptides—chal- Chem 8:1753–1764. lenges and perspectives for the development of novel anticancer drugs. Chem Phys 19. Albada HB, et al. (2012) Tuning the activity of short Arg-Trp antimicrobial peptides by Lipids 164(8):766–781. lipidation of C- or N-terminal lysine side-chain. ACS Med Chem Lett 3(12):980–984. 3. Mygind PH, et al. (2005) is a peptide antibiotic with therapeutic potential 20. Albada HB, et al. (2013) Short antibacterial peptides with significantly reduced he- from a saprophytic fungus. Nature 437(7061):975–980. molytic activity can be identified by a systematic L-to-D exchange scan of their amino 4. Schneider T, et al. (2010) Plectasin, a fungal defensin, targets the bacterial cell wall acid residues. ACS Comb Sci 15(11):585–592. precursor Lipid II. Science 328(5982):1168–1172. 21. Urban A, et al. (2007) Novel whole-cell antibiotic biosensors for compound discovery. 5. Patra M, Gasser G, Metzler-Nolte N (2012) Small organometallic compounds as anti- Appl Environ Microbiol 73(20):6436–6443. bacterial agents. Dalton Trans 41(21):6350–6358. 22. Brötz-Oesterhelt H, et al. (2005) Dysregulation of bacterial proteolytic machinery by 6. Pag U, et al. (2008) Analysis of in vitro activities and modes of action of synthetic anti- a new class of antibiotics. Nat Med 11(10):1082–1087. microbial peptides derived from an alpha-helical ‘sequence template’. J Antimicrob 23. Bandow JE (2005) Proteomic approaches to antibiotic drug discovery. Curr Protoc Chemother 61(2):341–352. Microbiol Chapter 1:2. 7. Zelezetsky I, Tossi A (2006) Alpha-helical antimicrobial peptides—using a sequence 24. Bandow JE, Brötz H, Leichert LI, Labischinski H, Hecker M (2003) Proteomic approach – template to guide structure-activity relationship studies. Biochim Biophys Acta to understanding antibiotic action. Antimicrob Agents Chemother 47(3):948 955. 25. Wenzel M, et al. (2011) Proteomic signature of biosynthesis inhibition 1758(9):1436–1449. 8. Zelezetsky I, et al. (2005) Controlled alteration of the shape and conformational available for in vivo mechanism-of-action studies. Antimicrob Agents Chemother 55(6):2590–2596. stability of alpha-helical cell-lytic peptides: Effect on mode of action and cell speci- 26. Wenzel M, et al. (2012) Proteomic response of Bacillus subtilis to lantibiotics reflects ficity. Biochem J 390(Pt 1):177–188. differences in interaction with the cytoplasmic membrane. Antimicrob Agents Che- 9. Rathinakumar R, Walkenhorst WF, Wimley WC (2009) Broad-spectrum antimicrobial mother 56(11):5749–5757. peptides by rational combinatorial design and high-throughput screening: The im- 27. Katsu T, et al. (1989) Mechanism of membrane damage induced by the amphipathic portance of interfacial activity. J Am Chem Soc 131(22):7609–7617. peptides gramicidin S and . Biochim Biophys Acta 983(2):135–141. 10. Sharma RK, Sundriyal S, Wangoo N, Tegge W, Jain R (2010) New antimicrobial hex- 28. Gross A, et al. (2012) A ruthenocene-PNA bioconjugate—synthesis, characterization, apeptides: Synthesis, antimicrobial activities, cytotoxicity, and mechanistic studies. cytotoxicity, and AAS-detected cellular uptake. Bioconjug Chem 23(9):1764–1774. ChemMedChem 5(1):86–95. 29. Matias VR, Beveridge TJ (2005) Cryo-electron microscopy reveals native polymeric cell 11. Wimley WC, Hristova K (2011) Antimicrobial peptides: Successes, challenges and un- wall structure in Bacillus subtilis 168 and the existence of a periplasmic space. Mol answered questions. J Membr Biol 239(1-2):27–34. Microbiol 56(1):240–251. 12. Yeaman MR, Yount NY (2003) Mechanisms of antimicrobial peptide action and re- 30. Matias VR, Beveridge TJ (2008) Lipoteichoic acid is a major component of the Bacillus sistance. Pharmacol Rev 55(1):27–55. subtilis periplasm. J Bacteriol 190(22):7414–7418. 13. Chan DI, Prenner EJ, Vogel HJ (2006) Tryptophan- and arginine-rich antimicrobial 31. Weidenmaier C, Peschel A (2008) Teichoic acids and related cell-wall glycopolymers in peptides: Structures and mechanisms of action. Biochim Biophys Acta 1758(9): Gram-positive physiology and host interactions. Nat Rev Microbiol 6(4):276–287. – 1184 1202. 32. Clejan S, Krulwich TA, Mondrus KR, Seto-Young D (1986) Membrane lipid compo- 14. Zweytick D, et al. (2011) Studies on lactoferricin-derived Escherichia coli membrane- sition of obligately and facultatively alkalophilic strains of Bacillus spp. JBacteriol active peptides reveal differences in the mechanism of N-acylated versus nonacylated 168(1):334–340. peptides. J Biol Chem 286(24):21266–21276. 33. Brisebois PP, Arnold AA, Chabre YM, Roy R, Marcotte I (2012) Comparative study of the 15. Zweytick D, Tumer S, Blondelle SE, Lohner K (2008) Membrane curvature stress interaction of fullerenol nanoparticles with eukaryotic and bacterial model membranes and antibacterial activity of lactoferricin derivatives. Biochem Biophys Res using solid-state NMR and FTIR spectroscopy. Eur Biophys J 41(6):535–544. Commun 369(2):395–400. 34. Duax WL, et al. (1996) Molecular structure and mechanisms of action of cyclic and 16. Chantson JT, Verga Falzacappa MV, Crovella S, Metzler-Nolte N (2006) Solid-phase linear ion transport antibiotics. Biopolymers 40(1):141–155. synthesis, characterization, and antibacterial activities of metallocene-peptide bio- 35. Strahl H, Hamoen LW (2010) Membrane potential is important for bacterial cell di- conjugates. ChemMedChem 1(11):1268–1274. vision. Proc Natl Acad Sci USA 107(27):12281–12286. 17. Strøm MB, et al. (2003) The pharmacophore of short cationic antibacterial peptides. 36. Andrés MT, Fierro JF (2010) Antimicrobial mechanism of action of transferrins: Se- + J Med Chem 46(9):1567–1570. lective inhibition of H -ATPase. Antimicrob Agents Chemother 54(10):4335–4342.

Wenzel et al. PNAS Early Edition | 9of10 Downloaded by guest on September 30, 2021 37. Mogi T, Ui H, Shiomi K, Omura S, Kita K (2008) Gramicidin S identified as a potent 64. Henry G, Deleu M, Jourdan E, Thonart P, Ongena M (2011) The bacterial lipopeptide inhibitor for cytochrome bd-type quinol oxidase. FEBS Lett 582(15):2299–2302. surfactin targets the lipid fraction of the plant plasma membrane to trigger immune- 38. Vonck J, Schäfer E (2009) Supramolecular organization of protein complexes in the related defence responses. Cell Microbiol 13(11):1824–1837. mitochondrial inner membrane. Biochim Biophys Acta 1793(1):117–124. 65. Desoignies N, Schramme F, Ongena M, Legrève A (2013) Systemic resistance induced 39. Hoffmann T, Boiangiu C, Moses S, Bremer E (2008) Responses of Bacillus subtilis to by Bacillus in Beta vulgaris reduces infection by the rhizomania disease hypotonic challenges: Physiological contributions of mechanosensitive channels to vector Polymyxa betae. Mol Plant Pathol 14(4):416–421. cellular survival. Appl Environ Microbiol 74(8):2454–2460. 66. Brotman Y, Makovitzki A, Shai Y, Chet I, Viterbo A (2009) Synthetic ultrashort cationic 40. Sitaram N, Nagaraj R (1999) Interaction of antimicrobial peptides with biological and lipopeptides induce systemic plant defense responses against bacterial and fungal model membranes: Structural and charge requirements for activity. Biochim Biophys pathogens. Appl Environ Microbiol 75(16):5373–5379. – Acta 1462(1-2):29 54. 67. Whatmore AM, Chudek JA, Reed RH (1990) The effects of osmotic upshock on the 41. Epand RM, et al. (2010) Lipid clustering by three homologous arginine-rich antimi- intracellular solute pools of Bacillus subtilis. J Gen Microbiol 136(12):2527–2535. crobial peptides is insensitive to amino acid arrangement and induced secondary 68. Hoffmann T, et al. (2012) Synthesis, release, and recapture of compatible solute – structure. Biochim Biophys Acta 1798(6):1272 1280. proline by osmotically stressed Bacillus subtilis cells. Appl Environ Microbiol 78(16): 42. Fuertes G, Giménez D, Esteban-Martín S, Sánchez-Muñoz OL, Salgado J (2011) A 5753–5762. lipocentric view of peptide-induced pores. Eur Biophys J 40(4):399–415. 69. Hoffmann T, et al. (2013) Osmotic control of opuA expression in Bacillus subtilis and its 43. Schibli DJ, Epand RF, Vogel HJ, Epand RM (2002) Tryptophan-rich antimicrobial modulation in response to intracellular glycine betaine and proline pools. J Bacteriol peptides: Comparative properties and membrane interactions. Biochem Cell Biol 195(3):510–522. 80(5):667–677. 70. Zaprasis A, et al. (2013) Osmoprotection of Bacillus subtilis through import and pro- 44. Rezansoff AJ, et al. (2005) Interactions of the antimicrobial peptide Ac-FRWWHR- teolysis of proline-containing peptides. Appl Environ Microbiol 79(2):576–587. NH( ) with model membrane systems and bacterial cells. J Pept Res 65(5):491–501. 2 71. Ajouz B, Berrier C, Garrigues A, Besnard M, Ghazi A (1998) Release of thioredoxin via 45. Scheinpflug K, Nikolenko H, Komarov IV, Rautenbach M, Dathe M (2013) What goes the mechanosensitive channel MscL during osmotic downshock of Escherichia coli around comes around-a comparative study of the influence of chemical modifications cells. J Biol Chem 273(41):26670–26674. on the antimicrobial properties of small cyclic peptides. Pharmaceuticals (Basel) 6(9): 72. Becker M, et al. (2013) Glutamate efflux mediated by Corynebacterium glutamicum 1130–1144. 46. Spindler EC, Hale JD, Giddings TH, Jr., Hancock RE, Gill RT (2011) Deciphering the MscCG, Escherichia coli MscS, and their derivatives. Biochim Biophys Acta 1828(4): – mode of action of the synthetic antimicrobial peptide Bac8c. Antimicrob Agents 1230 1240. Chemother 55(4):1706–1716. 73. Kouwen TR, et al. (2009) The large mechanosensitive channel MscL determines bac- 47. Mann PA, et al. (2013) Murgocil is a highly bioactive staphylococcal-specific inhibitor of terial susceptibility to the sublancin 168. Antimicrob Agents Chemother the glycosyltransferase enzyme MurG. ACS Chem Biol 8(11):2442–2451. 53(11):4702–4711. 48. Pogliano J, Pogliano N, Silverman JA (2012) Daptomycin-mediated reorganization of 74. Turner J, Cho Y, Dinh NN, Waring AJ, Lehrer RI (1998) Activities of LL-37, a cathelin- membrane architecture causes mislocalization of essential cell division proteins. associated antimicrobial peptide of human . Antimicrob Agents Chemother J Bacteriol 194(17):4494–4504. 42(9):2206–2214. 49. Bayer AS, Schneider T, Sahl H-G (2013) Mechanisms of daptomycin resistance in 75. Koo SP, Yeaman MR, Bayer AS (1996) Staphylocidal action of thrombin-induced : Role of the and cell wall. Ann N Y Acad Sci platelet microbicidal protein is influenced by microenvironment and target cell 1277:139–158. growth phase. Infect Immun 64(9):3758–3764. 50. Lenarcic R, et al. (2009) Localisation of DivIVA by targeting to negatively curved 76. Schneider T, et al. (2004) In vitro assembly of a complete, pentaglycine interpeptide membranes. EMBO J 28(15):2272–2282. bridge containing cell wall precursor (lipid II-Gly5) of Staphylococcus aureus. Mol 51. Wecke T, et al. (2009) Daptomycin versus Friulimicin B: In-depth profiling of Bacillus subtilis Microbiol 53(2):675–685. cell envelope stress responses. Antimicrob Agents Chemother 53(4):1619–1623. 77. Otto A, et al. (2010) Systems-wide temporal proteomic profiling in glucose-starved 52. Ha S, Walker D, Shi Y, Walker S (2000) The 1.9 A crystal structure of Escherichia coli Bacillus subtilis. Nat Commun 1:137. MurG, a membrane-associated glycosyltransferase involved in peptidoglycan bio- 78. Eymann C, et al. (2004) A comprehensive proteome map of growing Bacillus subtilis synthesis. Protein Sci 9(6):1045–1052. cells. Proteomics 4(10):2849–2876. 53. Bengtsson J, Tjalsma H, Rivolta C, Hederstedt L (1999) Subunit II of Bacillus subtilis 79. Wolff S, Hahne H, Hecker M, Becher D (2008) Complementary analysis of the vege- cytochrome c oxidase is a lipoprotein. J Bacteriol 181(2):685–688. tative membrane proteome of the human pathogen Staphylococcus aureus. Mol Cell 54. Bengtsson J, Rivolta C, Hederstedt L, Karamata D (1999) Bacillus subtilis contains two Proteomics 7(8):1460–1468. small c-type cytochromes with homologous heme domains but different types of 80. Vizcaíno JA, et al. (2013) The PRoteomics IDEntifications (PRIDE) database and asso- – membrane anchors. J Biol Chem 274(37):26179 26184. ciated tools: Status in 2013. Nucleic Acids Res 41(Database issue):D1063–D1069. 55. Fränzel B, et al. (2010) Corynebacterium glutamicum exhibits a membrane-related 81. Santhana Raj L, et al. (2007) Rapid method for transmission electron microscopy study response to a small ferrocene-conjugated antimicrobial peptide. JBiolInorgChem of Staphylococcus aureus ATCC 25923. Annals of Microscopy 7:102–108. – 15(8):1293 1303. 82. Gust R, et al. (1998) Stability and cellular studies of [rac-1,2-bis(4-fluorophenyl)- 56. López D, Kolter R (2010) Functional microdomains in bacterial membranes. Genes Dev ethylenediamine][cyclobutane-1,1- dicarboxylato]platinum(II), a novel, highly ac- 24(17):1893–1902. tive carboplatin derivative. JCancerResClinOncol124(11):585–597. 57. Kingston AW, Subramanian C, Rock CO, Helmann JD (2011) A σW-dependent stress 83. Schäfer S, Ott I, Gust R, Sheldrick WS (2007) Influence of the polypyridyl (pp) li- response in Bacillus subtilis that reduces membrane fluidity. Mol Microbiol 81(1): gand size on the DNA binding properties, cytotoxicity and cellular uptake of or- 69–79. ganoruthenium(II) complexes of the type [(η6-C Me )Ru(L)(pp)]n+. EurJInorg 58. Wolf D, et al. (2010) In-depth profiling of the LiaR response of Bacillus subtilis. 6 6 Chem 19:3034–3046. J Bacteriol 192(18):4680–4693. 84. Schatzschneider U, et al. (2008) Cellular uptake, cytotoxicity, and metabolic profiling 59. Kobayashi R, Suzuki T, Yoshida M (2007) Escherichia coli phage-shock protein A of human cells treated with ruthenium(II) polypyridyl complexes [Ru(bpy)2 (PspA) binds to membrane phospholipids and repairs proton leakage of the damaged — — = membranes. Mol Microbiol 66(1):100–109. (N N)]Cl2 with N N bpy, phen, dpq, dppz, and dppn. ChemMedChem 3(7): – 60. Bertsche U, et al. (2011) Correlation of daptomycin resistance in a clinical Staphylo- 1104 1109. coccus aureus strain with increased cell wall teichoic acid production and D-alanyla- 85. Burstein C, Tiankova L, Kepes A (1979) Respiratory control in Escherichia coli K 12. Eur – tion. Antimicrob Agents Chemother 55(8):3922–3928. J Biochem 94(2):387 392. 61. Rose WE, Fallon M, Moran JJ, Vanderloo JP (2012) Vancomycin tolerance in methi- 86. Palmgren MG, Sommarin M, Ulvskov P, Larsson C (1990) Effect of detergents on the (+) cillin-resistant Staphylococcus aureus: Influence of vancomycin, daptomycin, and H -ATPase activity of inside-out and right-side-out plant plasma membrane vesicles. on differential resistance gene expression. Antimicrob Agents Chemother Biochim Biophys Acta 1021(2):133–140. 56(8):4422–4427. 87. Smith JJ, McFeters GA (1997) Mechanisms of INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5- 62. Saar-Dover R, et al. (2012) D-alanylation of lipoteichoic acids confers resistance to phenyl tetrazolium chloride), and CTC (5-cyano-2,3-ditolyl tetrazolium chloride) re- cationic peptides in group B streptococcus by increasing the cell wall density. PLoS duction in Escherichia coli K-12. J Microbiol Methods 29(1):161–175. Pathog 8(9):e1002891. 88. Schmitt P, et al. (2010) Insight into invertebrate defensin mechanism of action: Iy- 63. Booth IR, Blount P (2012) The MscS and MscL families of mechanosensitive channels ster inhibit peptidoglycan biosynthesis by binding to lipid II. JBiolChem act as microbial emergency release valves. J Bacteriol 194(18):4802–4809. 285(38):29208–29216.

10 of 10 | www.pnas.org/cgi/doi/10.1073/pnas.1319900111 Wenzel et al. Downloaded by guest on September 30, 2021